The High-efficiency And Sensitive Detection Of Breast Cancer Molecular Marker CD44 And The Construction Of CD44-targeting Nanocomposite

Background:Breast cancer is one of the most common malignancies in women with increasing morbidity and mortality.The difficulty in treating breast cancer is due to the existence of breast cancer stem cells（BCSCs）,causing the metastasis and recurrence of the cancer.Cluster of differentiation 44（CD44）is one of the biomarkers of cancer stem cells.The expression level of CD44 is related to the malignant degree of breast cancer,and can be used as a target for breast cancer diagnosis and treatment.Objectives:To evaluate the grade malignancy and prognosis of breast cancer,the paper developed an electrochemical impedance biosensor for the determination of serum CD44concentration.To improve the therapeutic effect of these patients,the chemotherapeutic emodin-loaded pH-responsive and CD44-targeting nanocomposite was prepared and characterized,which laid the foundation for the diagnosis and treatment of breast cancer patients with high expression of CD44.Methods:This paper consists of two parts:the preparation of CD44-specific aptamer based electrochemical impedance biosensor for human serum CD44 detection;the construction and evaluation of the CD44-targeting and pH-responsive polymer micelles for targeted drug delivery to breast cancer cells.1 The thiol-modified CD44 aptamers were immobilized on the surface of the electrodes by gold-thiol interaction and the impedance increased.When incubated with different concentrations of CD44 solutions,the binding of CD44 to aptamers on gold electrodes led to the decrease of impedance,which was used to quantitative analyze of serum CD44 level.The biosensor was characterized by the electrochemical impedance spectroscopy.The experimental conditions were optimized.The calibration curve and the detection limit were determined.The anti-interference ability,stability and reproducibility of the sensor were evaluated.2 The biosensor was applied to the determination of serum CD44 levels of normal people and breast cancer patients.Pearson’s correlation analysis and Bland-Altman analysis were performed to evaluate the concordance of the results measured by the constructed biosensor and the ELISA kit.3 The amphiphilic block copolymer CD44p conjugated PEG-HES-PLA was synthesized by esterification and acetal reaction.The structure of the polymer was characterized by ¹H nuclear magnetic resonance spectra and X-ray photoelectron spectrometer.The critical micelle concentration value of the CD44p conjugated PEG-HES-PLA was determined by using pyrene as fluorescence probe.4 The emodin-loaded polymer micelle was prepared by the dialysis method.The particle size,zeta potential and morphology were characterized by laser particle size meter and transmission electron microscope.The standard curve of emodin was constructed by ultraviolet spectrophotometry and the determination of encapsulation efficiency and drug loading efficiency were based on the freeze- drying method.The stability of the polymer micelle was evaluated by the particles size change over 2 weeks in phosphate buffered saline（pH 7.4）at 4℃.The changes of polymer micelle size and zeta potential were measured at different pH solutions （pH 6.5,7.4）.In vitro emodin release of polymer micelles in different pH phosphate buffered saline（pH 6.5,7.4）was measured using the dialysis method.Results:1 Based on CD44-specific aptamers,the electrochemical impedance biosensor for serum CD44 detection was successfully constructed.The impedance decreased when the biosensor incubated with the target CD44.The biosensor differential responded to CD44 concentrations.The impedance decreased degree had a linear relationship with the logarithm of CD44 concentration at 0.1 ng/m L～1000.0 ng/m L.The limit of detection was 0.087 ng/m L（S/N=3）.The relative standard deviation was 3.96%（n=5）.The constructed biosensor has good anti-interference ability and stability.2 The concentrations of CD44 in human serum were measured by the constructed electrochemical impedance sensor and ELISA kit.Pearson correlation coefficient was 0.993,and the linear regression equation y=1.032x+6.121,R²=0.987.Bland-Altman analysis showed good consistency between two methods.The results showed that the serum CD44 level of breast cancer patients were 139.5±30.4ng/m L and the normal people CD44 level was 82.5±11.9 ng/m L.Compared with normal people,the breast cancer patients had higher serum CD44 level which was statistically significant tested by independent-samples T test（P<0.05）.The results suggested the potential of CD44 as diagnostic and prognostic biomarker of breast cancer.3 In this study,CD44 peptide conjugated amphiphilic triblock copolymer CD44p-PEG-HES-PLA was successfully synthesized.The critical micelle concentration was 1.3×10^-4 mg/m L.The particle size of the CD44-targeting and pH-responsive emodin-loaded polymer micelles was 154.5±0.9 nm and the zeta potential was-11.8±0.5 m V.The emodin-loaded polymer micelles were scattered,uniformly distributed spheres observed by transmission electron microscope.The encapsulation efficiency and drug loading efficiency were 98.5%and 9.6%,respectively.The emodin-loaded polymer micelles had good stability within 2 weeks in phosphate buffered saline（pH 7.4）at 4℃.When the CD44-targeting and pH-responsive emodin-loaded polymer micelles were in low pH phosphate buffered saline（pH 6.5）,the particle size decreased and zeta potential increased.The emodin release rate in PBS with pH at 6.5 was higher than that at pH 7.4,which proved the pH sensitivity.Conclusions:The CD44-specific aptamers based electrochemical impedance biosensor was successfully constructed for the convenient detection of human serum CD44.The CD44-targeting and pH-responsive emodin-loaded polymer micelles for breast cancer cell was successfully prepared with an appropriate particles size,the high drug loading capacity,good stability,and pH sensitivity,preparing for follow-up treatment for breast cancer.